The primary goal of this research project is to establish the genetic location of the genes encoding the A and the B subunits of cholera toxin. Nontoxinogenic (tox) mutants of V. cholerae will be isolated after insertion mutagenesis with the transposable elements, Tn5, Mud (Aplac) bacteriophage and two newly characterized, mutagenic cholera phages, VACI and VCAII. Tox- mutants possessing and insertion of a transposable element into a toxin structural gene will be identified by analyzing mutant DNA for toxin gene sequences by the Southern blotting method. Specific 32P- labelled DNA probes, derived from the cloned gene for the heat-labile enterotoxin of E. coli, will be used in these experiments to detect structural alterations in the cholera toxin A or B gene sequences. Tox- mutants shown to have an insertion in a toxin structural gene will then be used to map the position of the toxin gene by deletion analysis and conjugation. Regulatory mutants will also be identified and mapped by similar methods. Knowledge gained from these studies will have practical applications in the construction of toxin gene deletion mutants suitable for testing as live oral vaccines against cholera.